Large granular lymphocytic (LGL) leukemia is a rare lymphoproliferative disorder characterized by the presence of increased numbers of LGL cells in the peripheral blood.1 According to the World Health Organization (WHO) classification, LGL leukemia can be divided into the two morphologically similar subtypes of T-cell LGL leukemia and chronic natural killer (NK)-cell lymphoproliferative disorder (CLPD-NK).1 Although derived from distinct cell lineages, the clinical presentation is very similar and dominated by recurrent infections associated with neutropenia, anemia, splenomegaly and autoimmune diseases, particularly rheumatoid arthritis.2 Molecular diagnostics of these diseases thus far was limited to the analysis of T-cell receptor (TCR) rearrangements for evaluation of T-cell clonality. Very recently, somatic STAT3 mutations have been described with a high frequency of 40% in T-LGL leukemia3 and 30% in CLPD-NK.4 The discovery of STAT3 mutations in T-LGL leukemia reveals a significant diagnostic value as it allows with high specificity to distinguish many cases of LGL leukemia from other mature T-cell neoplasms and reactive conditions. Thus, it strongly supports immunophenotyping and morphology in diagnostics of T-LGL leukemia and CLPD-NK. All mutations were found to be located in the Src homology 2 (SH2) domain, which mediates the dimerization and activation of the STAT protein.3, 4 The aim of our study was to further analyze the frequency and potential prognostic impact of STAT3 mutations in patients with T-LGL leukemia in comparison with cases with other T-cell malignancies and reactive conditions. We analyzed 55 patients (49 peripheral blood samples, 6 bone-marrow samples) with T-LGL leukemia. Diagnosis of T-LGL leukemia was based on the following WHO criteria: a monoclonal TCR rearrangement, the presence of an abnormal cytotoxic T-cell population with expression of CD3, CD8 and CD57 detected by flow cytometry and an LGL count by peripheral blood smear of >2 × 109/l (however, cases with LGL <2 × 109/l that met all other criteria were counted as consistent with the diagnosis of T-LGL leukemia). Each patient met these criteria to be included into the study. The cohort was composed of 24 males and 31 females. Median age was 68.9 years (range: 30.3–84.3 years). For comparison, 79 cases with other T-cell malignancies were analyzed (61 with non-LGL mature T-cell neoplasms and 18 cases with T-ALL; median age: 64.3 years; range: 19.1–86.2 years). Furthermore, 39 cases with reactive conditions were examined. Samples of these patients were referred to our lab with suspected mature T-cell neoplasm, which was excluded by cytomorphology and immunophenotyping (median age: 56.9 years; range: 21.0–87.6 years). All patients gave an informed consent for scientific evaluations, for example, molecular studies. The study was approved by the Internal Review Board of the MLL Munich Leukemia Laboratory and adhered to the tenets of the Declaration of Helsinki. Cytomorphological assessment was based on May–Grünwald–Giemsa stains. Immunophenotyping was performed in 43/55 cases according to the previously described procedures.5 The antigens analyzed included CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD34, CD56, CD57, TCR α/β, TCR γ/δ and TdT; antibodies were purchased from Immunotech (Marseilles, France). Screening for STAT3 mutations was performed by direct Sanger sequencing of the SH2 domain that is encoded by exon 21 as previously described.3 TCR rearrangements were assessed by multiplex PCR with subsequent fragment analysis.6 Dichotomous variables were compared between different groups using the χ2-test and continuous variables by Student’s t-test. Results were considered significant at P<0.05. All the reported P-values are two-sided. No adjustments for multiple comparisons were performed. SPSS version 19.0 (IBM Corporation, Armonk, NY, USA) was used for statistical analysis. Overall, in 40/55 T-LGL leukemia cases (72.7%), 41 STAT3 mutations were detected. All mutations were missense mutations. Six different mutations were observed (Figure 1). Tyr640Phe (n=18) and Asp661Tyr (n=13) accounted for 75.6% of all mutations detected. One patient harbored two mutations (Asn647Ile and Tyr640Phe). We also detected a Ile659Leu in one patient, which has not been described previously.3, 4 There was no association of STAT3 mutations with age, sex and peripheral blood counts in T-LGL leukemia cases (Table 1). Cases with STAT3 mutations had a stronger expression of CD3 (percentage positive cells, 76±20% vs 58±22%, P=0.01) and TCRαβ (68±19% vs 48±20%, P=0.004). This is probably explained by the significantly elevated clonal size in STAT3-mutated cases compared with STAT3 wild-type cases (percentage positive cells, 31±16% vs 16±13%, P=0.006). Furthermore, we correlated STAT3 mutation load to clonal size as evaluated by immunophenotyping by Spearman’s rank correlation and found a significant positive correlation (correlation coefficient=0.573, P<0.001). However, the detection of STAT3 mutations was not limited by the size of the pathological T-cell clone, as we were able to detect STAT3 mutations in clones as small as 6% as determined by immunophenotyping. Furthermore, the cohort also contained patients with clonal expansion of T-LGL cells (clonal size up to 73%) that were negative for STAT3 mutations. In addition, we validated nine STAT3 unmutated samples with pathological T-cell clones sizes of 1%–15% by a more sensitive next-generation sequencing approach (Roche 454, Roche Applied Science, Branford, CT, USA) and discovered only one additional STAT3-mutated patient. This patient had a T-cell clone size of 10% and a STAT3 mutation load of 3%. TCR gene usage did not differ between the STAT3-mutated and unmutated cases.
In addition to the T-LGL cases, also seven patients with CLPD-NK were analyzed. A STAT3 mutation was detected in 3/7 cases. One patient had a duplication of one amino acid (Tyr657dup), a mutation so far not observed in T-LGL leukemias (Figure 1). However, owing to the limited number of CLPD-NK cases overall, this data needs further confirmation. No STAT3 mutation was identified in a cohort of 118 cases diagnosed with non-LGL mature T-cell neoplasms, T-ALL or reactive conditions. Thus, STAT3 mutations appear to provide a highly specific marker for T-LGL and CLPD-NK leukemias.
We were also interested in whether STAT3 mutations were of any prognostic impact for T-LGL leukemia patients. However, owing to limited follow-up information and the generally good prognosis of LGL leukemia,7 survival analysis was not informative, as all patients were alive at the time of the present study. Of 11 patients, blood or bone-marrow samples were available at different time points during clinical course. Ten out of eleven patients showed a clinical course with comparable STAT3 mutation load and correlating TCR rearrangement status. One out of eleven patient with a Tyr640Phe mutation in STAT3 showed spontaneous remission of T-LGL leukemia at the molecular level without having received any therapy. Remission was also confirmed by immunophenotyping and cytomorphology. Spontaneous remission of T-LGL has previously been reported,8, 9 however, to our knowledge, this is the first case of spontaneous remission in STAT3-mutated T-LGL leukemia. Importantly, T-NHL often have an undulating course of disease and the STAT3-mutated clone may have decreased to a non-detectable size.
In conclusion, we were able to confirm the presence of STAT3 mutations in T-LGL as published by Koskela et al.,3 with an even higher frequency of 72.7%. This might be explained by the fact that Koskela et al.3 included only patients with one major Vβ TCR clone to ensure that the cohort is clinically and genetically uniform. In contrast, our cohort consisted of patients with unselected TCR rearrangements. We show that the discovery of STAT3 mutations in T-LGL leukemia has significant diagnostic value as it allows with high specificity to distinguish T-LGL leukemia from other mature T-cell neoplasms and reactive conditions. This strongly supports immunophenotyping and morphology in diagnosing T-LGL leukemia.
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We thank all clinicians for sending samples to our laboratory for diagnostic purposes, and for providing clinical information and follow-up data. In addition, we would like to thank all co-workers at the MLL Munich Leukemia Laboratory for approaching together many aspects in the field of leukemia diagnostics and research. In addition, we are grateful for the data management support performed by Tamara Alpermann.
WK, CH, TH and SS are part owners of the MLL Munich Leukemia Laboratory. AF and VG are employed by the MLL Munich Leukemia Laboratory.
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Fasan, A., Kern, W., Grossmann, V. et al. STAT3 mutations are highly specific for large granular lymphocytic leukemia. Leukemia 27, 1598–1600 (2013). https://doi.org/10.1038/leu.2012.350
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